1. Field of the Invention
The present invention relates generally to the translocation of proteins and other compounds into and out of the nucleus of cells and, more particularly, relates to novel nuclear localization signal sequences and nuclear export signal sequences and their uses, such as, for example and without limitation, regulation of nucleic acid expression, transfection of eukaryotic cells, gene therapy, protection from toxic chemicals, transport of anti-cancer agents, etc.
2. Description of Related Art
The exchange of macromolecules between the cytoplasm and the cell nucleus is a basic biological process in eukaryotic cells central to the regulation of gene expression (which underlies all aspects of development, morphogenesis, and signaling pathways in eukaryotic organisms). Nuclear traffic occurs exclusively through the nuclear pore complex (NPC), a huge multi-proteic complex which lies across the nuclear membrane. While small molecules (up to 40-60 kDa) can diffuse through the NPC, nuclear import of larger molecules, such as proteins, is mediated by specific nuclear localization signal (NLS) sequences contained either in the transported molecule (Garcia-Bustos et al., Biochim. Biophys. Acta 1071:83-101, 1991) or contained in a shuttle protein which binds to the protein being transported.
NLS sequences typically are small, mostly basic, amino acid sequences which can be classified into three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO: 22) (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (KR)OCXXXXXXXXKKKL) (SEQ. ID NO: 23) and (iii) noncanonical sequences such as M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481, 1991).
The steps involved in the import mechanism of proteins into eukaryotic nuclei have been elucidated (Nigg, E. A., Nature, 386:779-87, 1997; Gorlich, D., EMBO J., 17:2721-7, 1998). To be transported, the NLS sequence is recognized by members of the importin family of proteins (also referred to as karyopherins), which then act as carriers to transport the substrate protein across the NPC. Inside the nucleus, the importin-substrate complex dissociates, liberating the substrate protein, and the importin carrier ultimately returns to the cytoplasm. The small GTPase Ran plays a pivotal role in this process by promoting, in its GTP-bound form, the dissociation of the import complex and the subsequent recycling of the importin carrier.
Once in the nucleus, many proteins are transported back to the cytoplasm as an essential step in their biological function. The export of macromolecules from the nucleus also relies on the existence of a specific signal in the substrate to be exported. For example, the Rev protein of human immunodeficiency virus type 1 (HIV-1) exits the nucleus, facilitating export of the unspliced viral RNA (Pollard and Malim, Ann. Rev. Microbiol., 52:491-532, 1998). Rev protein nuclear export is mediated by a specific nuclear export signal (NES) sequence consisting of the leucine-rich sequence, LPPLERLTL (SEO ID NO: 24), found also in proteins of other viruses (Dobbeistein et al., EMBO J. 16:4276-4284, 1997). Additionally, numerous cellular proteins, such as I-KB and MAPKK, contain potential NES sequences that may regulate the biological activity of these proteins by controlling their nuclear export (Ulinian et al., Cell 90:967-970, 1997). Known NES sequences essentially are short, leucine-rich, hydrophobic peptide motifs which mediate the handling of the substrate by other members of the importin β family of proteins, called exportins. Nuclear import and export processes thus are tightly linked.
The relatively small size of the NLS and NES sequences and, more importantly, the lack of clear and consistent consensus motifs in these signals, make it difficult to predict their presence in a given protein based solely on the analysis of its amino acid sequence. Furthermore, even if a consensus NLS or NES is found, it may not represent a functional signal. For example, β-glucuronidase (GUS), a commonly-used reporter enzyme which resides exclusively in the cell cytoplasm, carries a perfect, albeit non-functional, bipartite NLS sequence at its carboxy terminus. The only practical way to identify active NLS or NES sequences is by microinjecting (Guralnick et al., Plant Cell 8:363-373, 1996) or expressing the protein of interest in eukaryotic cells (Varagona et al., Plant Cell 3:105-113, 1991), heterokaryon formation (Michael et al., Cell 83:415-422, 1995), or using an in vitro transport system (Ossareh-Nazari et al., Science, 278:141-144, 1997).
A need exists, therefore, for determining new and unique NLS sequences which can translocate proteins and other compounds effectively and efficiently into or out of the cell nucleus.